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| Status |
Public on Oct 16, 2014 |
| Title |
H3 ChIP-seq cycle 14c |
| Sample type |
SRA |
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| Source name |
embryos, cycle 14c, H3 ChIP
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain/background: Oregon R
genotype/variation: wild-type
tissue: embryo
developmental stage: mitotic cycle 14(c-d)
chip antibody: anti-H3 (Abcam, 1791, lot GR65697-1)
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| Treatment protocol |
Embryos were collected for 30 minutes, and then allowed to develop for 55, 85, 120 or 160 additional minutes before being harvested and fixed with formaldehyde. The fixed embryos were hand sorted in small batches using an inverted microscope to remove embryos younger or older than the targeted age range based on morphology of the embryos as previously described (Harrison et al., 2011). After sorting, embryos were stored at -80oC. After all collections were completed, the sorted embryos of each stage were pooled, and a sample of each pool were stained with DAPI. The ages of the embryos and their distribution in the two younger embryo pools (c7-9, and c11-13) were determined based on nuclei density of the stained embryos. The ages of embryos between c14a and c14c, both of which were distinct from c13 based on nuclei density, were determined based on morphology. For the ChIP-seq experiment in zelda mutant embryos, for which wild type (wt) Oregon R embryos were assayed in parallel as control, the flies were allowed to lay eggs for 3 hours and the embryos were aged for one additional hour before being treated with formaldehyde.
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| Growth protocol |
D. melanogaster flies were maintained in large population cages in an incubator set at standard conditions (25°C).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
7.5, 0.7, 0.4, and 0.3 g of embryos at four different stages, respectively, were used to prepare chromatin for immunoprecipitation following the CsCl2 gradient ultracentrifugation protocol as previously described (Harrison et al., 2011).The chromatin obtained was fragmented to sizes ranging from 100 to 300 bp using a Bioruptor (Diagenode, Inc.) for a total processing time of 140 min (15 s on, 45 s off), with power setting at "H". Prior to carrying out chromatin immunoprecipitation, we mixed the chromatin from each sample with a roughly equivalent amount of chromatin isolated from stage 5 (mitotic cycle 14) D. pseudoobscura embryos, and used about 2 µg of total chromatin (1 µg each of the D. melanogaster and D. pseudoobscura chromatin) for each chromatin immunoprecipitation. The chromatin immunoprecipitation reactions were carried out as described previously (Harrison et al., 2011) with 0.5 ug anti-H4K5ac (Millipore, 07-327), 0.5 ug anti-H3K4me3 (Abcam, ab8580), 0.5 ug anti-H3K27ac (Abcam, ab4729), 1 ug anti-H3 (Abcam, ab1791), 0.75 ug anti-H3K4me1 (Abcam, ab8895), 0.75 ug anti-H4K8ac (Abcam, ab15823), 1.5 ul anti-H3K9ac (Activemotif, 39138), 0.75 ug anti-H3K18ac (Abcam, ab1191), 3 ug anti-H3K27me3 (Millipore, 07-449), or 0.75 ug anti-H3K36me3 (Abcam, ab9050).
The sequencing libraries were prepared from the ChIP and Input DNA samples using the Illumina TruSeq DNA Sample Preparation kit following the manufacturer's instructions, and DNA was subjected to ultra-high-throughput sequencing on Illumina HiSeq 2000 DNA sequencers.
The libraries with different indexes were usually combined into pools, and each library pool was sequenced (100 bp single-end read) in a single lane in a flow cell.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Sample name: Dmel-H3-c14c
ChIP for histone H3 of wt Dmel embryo (Oregon R) at mitotic cycles 14c-d (hand sorted).
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| Data processing |
Basecalls were performed using CASAVA_v1.8.0.
Sequenced reads were mapped jointly to the April 2006 assembly of the D. melanogaster genome [FlyBase Release 5] and the November 2004 assembly of the D. pseudoobscura genome [FlyBase Release 1.0] using Bowtie (Langmead, 2010) with the command-line options "-q -5 5 -3 5 -l 70 -n 2 -a -m 1 -best -strata", thereby trimming 5 bases from each end of the 100 base single reads, and keeping only tags that mapped uniquely to the genomes with at most two mismatches.
We called peaks for each experiment using MACS (Zhang et al., 2008) v1.4.2 with the options "-- nomodel --shiftsize=130", and used Input as controls.
Genome_build: Drosophila melanogaster FlyBase Release 5 (April 2006).
Genome_build: Drosophila pseudoobscura FlyBase Release 1.0 (November 2004).
Supplementary_files_format_and_content: wig files were generated by extending each aligned read based on the average size of the DNA fragments in the libraries, and the values at each position were further normalized based on the number of D. pseudoobscura reads from each sequenced sample, which served as internal control for the ChIP-seq experiments. The peak files were generated from the MACS peak call program (Zhang et al., 2008).
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| Submission date |
Jul 02, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael B Eisen |
| E-mail(s) |
[email protected]
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| Organization name |
HHMI/Univ. California, Berkeley
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| Department |
MCB
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| Lab |
Mike Eisen
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| Street address |
379 Stanley Hall, MC 3220
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| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE58935 |
Establishment of regions of genomic activity during the Drosophila maternal-to-zygotic transition |
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| Relations |
| BioSample |
SAMN02900711 |
| SRA |
SRX645136 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1424925_Dmel-H3-c14c.wig.gz |
30.5 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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